Regulation of brain aging through epigenetic processes is currently one of the most provocative areas of aging research. Epigenetic processes in the central nervous system may play a mechanistic role in susceptibility to and progression of cognitive decline and age-related neurodegenerative disease such as Alzheimer?s disease and other dementias. DNA modifications, principally methylation and hydroxymethylation of cytosines (mC and hmC respectively), are fundamental regulators of DNA accessibility and gene regulation/expression with differential effects on gene expression depending on the modification (mC/hmC), context CG/CH, and genomic location. A barrier to progress in understanding the role of epigenetic mechanisms in brain aging, and DNA modifications in particular, has been the lack of quantitatively accurate, genome-wide data. Without the knowledge of the specific genomic locations of altered modifications with aging and it is impossible to design well-rationalized, mechanistic studies that unravel the functional effects of epigenetic changes. Therefore, the critical next step for the field is to generate this genome-wide data of mC and hmC in CG and CH contexts in specific cell types and in both males and females. To address this barrier to progress we have developed innovative methods to analyze mC and hmC levels across the genome with absolute quantitation in a base- and strand-specific manner. Using these novel tools, we have found that there are significant changes with aging in the patterns of hippocampal mC and hmC, that these changes are principally sex-specific, and they correspond to altered gene expression. Intriguingly, we have also identified non-CpG methylation as the primary form of altered methylation with aging and a male-specific increased inter-animal variance (methylation entropy) across the genome with aging in the hippocampus. These findings raise significant questions that must be addressed to move the field to mechanistic studies. Is the neuroepigenome altered in a similar or dissimilar manner across CNS cell types and between sexes? Can age-related changes in the neuroepigenome be prevented? What regions of the genome should be targeted by epigenome editing approaches to test whether brain aging can be prevented or reversed by maintaining or restoring a ?youthful? DNA modification pattern? In Aim 1, cell type-specific changes in mC/hmC with aging in the CNS of male and female mice will be examined by whole genome sequencing (WGoxBS). Microglia, astrocytes, and neurons isolated from the hippocampus by both cell surface markers and NuTRAP technology will be examined. Young (3M), Adult (12M), and Aged (24M) C57Bl6 male and female mice will be examined and mC/hmC data will be concatenated with paired RNA-Seq data. Bioinformatic approaches will then be used to determine the role of altered modification patterns in age-related changes in gene expression, enrichment of differential modifications in regulatory regions of the genome, and to identify genomic loci for epigenome editing. In Aim 2 the ability of caloric restriction to prevent age-related changes in DNA methylation and hydroxymethylation and maintain a ?young? neuroepigenome will be determined in neurons, astrocytes, and microglia. At the tissue level, we have found prevention of age-related epigenomic changes by caloric restriction in males but the effects on isolated cell populations and females are unknown. Using a unique database of all publicly available, annotated human methylation data we will validate aspects of our finding in humans. These studies will allow the determination of critical genomic regions with altered DNA modification patterns that can be manipulated in future interventional studies. The ultimate goal of the research being clinical interventions that target the epigenome to maintain brain function with aging and prevent age-related neurological disease in Veterans.